EI and CI GC Ms

Analysis of
Pharmacologically
Relevant Compounds
using
GC/MSD EI/PCI/NCI
Compendium of Applications
Analysis of
Pharmacologically Relevant Compounds
using
GC/MSD EI/PCI/NCI
Compendium of Applications
Preface
The combination of gas
chromatography with mass
spectrometry has been
successfully used for decades.
The now standard and universal
technique of electron impact
ionization (EI) has provided,
with respect to sensitivity and
compound identification,
extraordinary performance and
the volume of spectral reference
data has continued to increase
over the years. Commercially
available mass spectral libraries
now contain more than 350,000
entries. Instrument sensitivity
has improved such that sufficient
sensitivity is available in full
scan mode for determinations
at analyte concentrations in
the range of 10pg/l to 1pg/l.
However the EI technique still
leaves some analytical demands
unfulfilled. For example, if the
EI spectrum shows little or no
definitive information about
the analyte molecule, such as
molecular weight, or, in samples
where matrix interferences
convolute the spectral
information due to insufficient
selectivity, then successful
detection can decrease
drastically.
For such cases, chemical
ionization techniques (CI) are
available which enhance and
extend the use of mass
spectrometry. The positive
chemical ionization (PCI) mode
can usually lead to molecular
weight information since
protonated and adduct species of
sufficient intensity can provide
distinctive information about the
correct molecular mass. For

compounds with high electron
affinity, the electron capture
negative ion chemical ionization
(ECNI or NCI) mode offers
selectivity and the utmost
sensitivity even in difficult
matrices. Detection limits in
the femtogram range can often
be easily achieved. Stable
instrumental parameters can
insure high reproducibility over
a wide concentration range.
Importantly, the new instrumental
platforms provide user-friendly
CI operational features, such as
autotuning, that make CI similar
to EI in convenience.
With CI techniques, mass
spectrometry becomes a
powerful, unique, problem-solving
tool. CI is the most creative
MS technique. After becoming
familiar with some criteria and
the effects of certain instrumental
parameters, the user will rapidly
achieve positive results, and the
usefulness and fascination of CI
will become readily apparent.
To assist and convey such an
experience is the aim of this
compendium.
I would like to thank
Dr. Harry Prest, Senior
Application Chemist,
Agilent Technologies,
Chemical Solution Business
Division, Palo Alto, USA,
for his helpful comments
and advice in preparing this
applications compendium.
H.-Jrgen Schulz
February, 2002
1. Introduction
1.1 Distinctions
between EI &CI
2. Positive Chemical
Ionization (PCI)
2.1 Ion Source Configuration
2.2 PCI Reactions of
different Reagent Gases
2.3 Examples of EI and PCI
3. Electron Capture Negative
Ionization (ECNI)
3.1 Optimizing ECNI Analysis
4. Practical Operating
Advice
4.1 Gas Chromatography
4.2 PCI & NCI Conditions
intermediate chemical agent.

This is particularly true in positive
chemical ionization (PCI). In PCI,
the ion source is filled with a
reagent gas which is ionized to
create reagent ions which react
with the analyte. The interesting
reaction products are positive
ions which are collected and
measured in PCI. Negative ions
are also formed by this chemical
process and this is a form of NCI.
The gas filling the source can also
be used to buffer or thermalize
electrons. These slow electrons
can be captured very efficiently
by analyte molecules to form
negative ions which is called
electron capture negative
ionization (ECNI). In casual
usage, when the resulting ions
being measured are positive,
it is referred to as PCI and when
negative, NCI.
4.3 Derivatization
5. Instrumentation
6. Literature
1. Introduction
The technique of chemical
ionization mass spectrometry
(CIMS) was pioneered by Munson
and Field in 1966. CIMS can
be considered an alternative
ionization approach to electron
impact (EI) and offers the
opportunity to determine the
molecular weight of the analytes
and in selected cases, CIMS
measurements show very high
selectivity and outstanding
sensitivity.
1.1 Distinctions between EI & CI
In EI mode, relatively high energy
electrons (70 eV) collide with
analyte molecules producing
positive ions and other species.
The fragmentation process,
executed under constant
conditions, is well understood
and the positive ion fragmentation
pattern, which is the EI mass
spectrum of the analyte, is used
for compound identification.
Whereas EI is a direct energy
transfer process with electron
kinetic energy deposited directly
in an analyte molecule, CI is an
indirect process involving an
2. Positive Chemical Ionization (PCI)

PCI mode is preferred in cases
that the EI mass spectrum
contains mostly low mass-to
charge ratio fragments and
therefore little or no information
about the molecular weight of
the analyte. The success of the
technique is strongly dependent
on the choice of the applied
reagent gas. The literature
documents a multitude of
examples for structure
elucidations with PCI techniques
by using different reagent gases,
in addition to the elementary
use of PCI for molecular weight.
PCI sensitivity is comparable
to EI sensitivity but can offer
significant improvements in
actual samples depending on
the choice of CI reagent gas and
nature of the interferences. In
terms of the signal-to-noise of the
base peaks, PCI often provides a
better result than EI and therefore
is well suited to selected ion
monitoring (SIM) acquisitions.
2.1 Ion Source Configuration
The CI ion source resembles
the EI source but is designed
to have an ionization chamber
(< 1ml volume) where the
reactions take place that is much
more enclosed. The filament
generating the electrons is
1
positioned just outside the

chamber. The electrons emitted
from the filament into the ion
chamber are accelerated to
between 100 and 200 eV for
optimal penetration of the reagent
gas. The electron entrance orifice
is small to keep the chamber tight
and reagent gas pressure high.
The reagent gas enters the
ionization chamber via the
GC/MSD interface. Because the
amount of reagent gas in the
source completely overwhelms
the analyte, the reaction of the
analyte is very efficient. The shape
of the source is designed that the
partial pressure in the outer
chamber is about 10-5 to 10-6 torr
in order to maintain a collision free
path for the ions to reach the
analyser. Mass spectrometry using
such a source design is named as
High Pressure Mass Spectrometry
(HPMS).
2.2 PCI reactions of different
Reagent Gases
The most frequently used reagent
gases are methane, iso-butane and
ammonia. The reagent gas is
ionized by electrons entering the
ionization source and, because
the pressure of the reagent gas is
high, a number of reactions occur.
The principal methane reactions
are:
CH4 + e- CH4+, CH3+, CH2+.
CH4 +CH4+ CH5+ + CH3. mz 17
CH2+. +CH4 C2H4+. + H2 mz 28
CH2+. +CH4 C2H3+ +H2+H.mz 27
CH3+ +CH4 C2H5+ + H2 mz 29
C2H3+ +CH4 C3H5+ + H2 mz 41
This stepwise description of the
gas phase reactions is for clarity
only and in fact all the processes
are proceeding simultaneously.
Of special interest is the CH5+ ion
which is a strong proton donator
and can form a protonated
molecule with an analyte.
Additionally methane forms
characteristic molecular adduct
ions :
CH5+ +M [MH]+ + CH4 mz M+1
C2H5+ +M [M+C2H5]+ mz M+29
C3H5+ +M [M+C3H5]+ mz M+41
Such processes are described by
the term Proton Affinity (PA). PA
is defined as the thermochemical
ability of the reaction partners to

transfer protons. In order to
generate a protonated molecule,
the PA of the analyte must be
greater than that of the reagent
gas ion. As the difference
between the PA of the analyte
and the PA of the reagent gas
ion increases the amount of
fragmentation of the analyte
increases. Referring to literature,
the PA of organic compounds is
between 180kcal/mol to
240kcal/mol. In most cases, the
PA of the compound of interest is
unknown and successful PCI
requires experimentation with
reagent gases in each case.
Reagent Gas Ions and their
Proton Affinities (kcal/mol):
Hydrogen
Methane
iso-Butane
Ammonia
H3+
CH5+
C4H9+
NH4+
The choice of the applied reagent

gas determines the fragmentation
behavior of the analyte and
consequently the result of the
PCI measurement. PCI processes
generating little fragmentation
are referred to as soft ionization
and the corresponding the reagent
gases are called soft reagent gas.
Conversely, reactions creating a
great deal of fragmentation are
named hard ionization and the
gas, a hard reagent gas .
In some cases the Hydride
Abstraction Reaction is of
interest, and often occurs with
long chain alkanes or compounds
containing long chain alkyl
groups:
CH5+ + M (M-H)+
In these cases the PA of the
reagent gas ion is greater than
the PA of the analyte.
2.3 Examples of EI/PCI
Figure 1 shows an example of the
differences in response for the
different ionization processes and
PCI reagent gases for methyl
palmitate. In full scan mode
the total ion response follows
EI > PCI/CH4 > PCI/NH3.
The spectra reflects the
characteristics of each reaction,
Figure 2
101
132
196
204
M+ = 270 m/z
Ammonia has a high PA value

which is closest of the common
gases to the PA of most organic
molecules so it often provides
better differentiation (more
selectively) between the sample
matrix and the analyte. Non-polar
compounds or matrices consisting
mostly of C and H, are less readily
ionized in comparison to more
polar compounds. For example,
carboxylic esters, such as the
phthalates (Agilent Technologies
Application Note 5988-2244EN)
show a high affinity for ammonia
because of the polar ester-linkages
(COO groups).
[M-H]+ = 269 m/z

[M+H]+ = 271 m/z
[M+C2H5]+ = 299 m/z
[M+C3H5]+ = 311 m/z
Formation of Ammonia
Reagent Gas Ions:
NH3 + e- NH3+, NH2+
NH3+ +NH3NH4+ + NH2+ mz 18
NH2+ +NH3NH3+ + NH2 mz 17
(NH4NH3)+ mz 35
(NH4(NH3)2)+mz 52
[M+NH4]+ = 288 m/z
Typical ammonia reactions

with an analyte (M)
M + NH4+ (M+NH4)+ M + 18
M + NH4+ (M+H)+ +NH3 M + 1
Analytes suited for ammonia
reaction generate the characteristic
[M+NH4]+ adduct ion, some times
the protonated molecule and most
often both.
Figure 1. Total ion chromatograms of

methyl palmitate at 600pg injected, in EI (top),
PCI-CH4 (middle) and PCI-NH3 (bottom)
Figure 2. Full Scan Spectra referring

to Figure 1
3. Electron Capture Negative

Ionization (ECNI)
The importance of the ECNI
technique becomes readily
evident in view of the selectivity
of the detection of suitable
analytes at very low concentration
levels (ppt). In addition, the typical
sample matrix interferences are
generally suppressed in ECNI MS
mode resulting in very high signalto-noise values. Suitable analytes
for NCI, have a high electron
capture capacity or high electron
affinity (EA). The expression
chemical ionization is not
applicable because the important
part of the reaction is achieved by
capture of low energy electrons
thermal electrons by the
analyte so there are no ion
molecular reactions involved in
the initial ion creation process.
Thermal electrons are generated
by collision of electrons emitted
from the filament with buffer gas
(e.g. methane) molecules located
at high pressure in the ionization
chamber of the source:
e**- (70 eV) + CH4 (buffer gas)
e*- thermal Electrons (2 eV)
e*- + M MAnalytes with high EA form stable
molecular anions, M-, and show a
simple spectrum. (See Figure 3).
This process is resonant electron
capture and commonly called
NCI. Electrons with an energy
potential of about 15 eV lead to
dissociative reactions:
e* (15eV) + MX M + XMass spectra resulting from
these reactions exhibit more
fragmentation and less sensitivity
compared with the ECNI process.
Though it is hard to predict which
analytes are suitable for NCI some
rules of thumb are used. Good
results are often obtained with
compounds already successfully
analyzed by GC/ECD. ECNI
candidates contain multiple
halogen groups, or nitro groups,
or double bond and/or conjugated
structures, and/or hetero atoms.
Polar compounds which are
suitable for derivatization can
be modified with perfluoro
reagents in order to integrate a
group with a high EA into the
consequently, the sensitivity. Low

ion source temperature favors the
electron capture process. As a
consequence of the operation of
the source (hot filament) and the
GC (hot carrier gas), the lowest
practical source temperature is
about 150C. Considering the
analyte elution temperature, the
ion source body sometimes is a
cold spot and might be a reason
for tailing peak shape and matrix
effects.
Figure 3. Mass spectra of tetrahydrocannabinol

as the trifluoroacetic acid anhydride
derivative (nominal mol. wt. 410 g/mol)
in EI (top) and ECNI-CH4 (bottom)
molecular structure and improve

chromatography.
3.1 Optimizing ECNI measurements
In ECNI mode some ion source
operating parameters can be
modified to improve sensitivity.
Buffer Gas
Raising the flow amount of the
buffer gas, which influences the
collision process, will sometimes
lead to an increase in response.
Such optimization refers to the
term High Pressure Electron
Capture Mass Spectrometry
(HPECMS). The quality and purity
of the buffer gas are of importance.
The gas purity should be in the
range of 99.95% (3.5) to 99.995%
(4.5). In addition to methane,
ammonia and carbon dioxide are
frequently applied. Oxygen and
water are very efficient at collecting
electrons and can suppress analyte
response and great care must be
taken that they are be excluded.
Ion Source Temperature
The ionization chamber
temperature dramatically affects
the reaction yield and the
fragmentation behavior and
3
Tuning Parameters
The tuning program controls the
parameters of electron energy
(eV) and emission current (A).
Electron energy (EE) has some
influence on the mobility of the
electrons and their penetration
efficiency in the ionization
chamber. A high EE value is
usually advantageous. Emission
current (EC) is related to the
amount of electrons emitting from
the filament. Increasing the EC
value leads to response additional
but filament lifetime has to be
considered. Higher ECs lower
filament life and the accuracy
of the filament position above
the electron entrance slit may
become degraded due to
deformation.
4. Practical Hints
The following hints are based
on the experience of the author
and are to be considered as
advice and do not represent a
guarantee in all circumstances
or applications.
4.1 Gas Chromatography
Chemical Ionization requires the
same GC criteria as applied in
EI mode. Carrier gas purifiers
are highly recommended. Also
an air-water check should be
performed prior to switching to CI.
Before developing a CI method,
all GC operating parameters such
as injection technique and the
capillary column should be tested
in EI mode in order to avoid any
kind of sample discrimination.
Thermally labile compounds are
analysed using on-column or PTV
injection systems which permit
cryo-focussing and sample
enrichment. Splitless injection
executed in pulsed-pressure mode
has the advantages of almost

quantitative sample transfer from
the injection liner into the column
and the reduced residence time of
the sample in the injector inlet.
The liner type has to be appropriate
to the injection technique. In this
compendium, for splitless injection
a deactivated double-taper liner
(Agilent Part Nr. 5181-3315) was
used. The choice of the column is
always related to the analytical
problem, however, for the majority
of the analytes documented
here, the MSD standard column
(HP-5ms, 30m x 0.25mm x 0.25m,
Agilent Part Nr. 19091S-433)
was adequate. Sample matrix
interferences may suggest use
of another phase and /or GC
oven program.
to the literature references and to

the authors lab experiences.
Derivatization reagents and
reaction (incubation) criteria are
matter of choice and may be
varied in order to improve
reaction yield and sensitivity.
For a reaction vial, the High
Recovery Vial, 1.5ml volume,
conical bottom autosampler vial
(Agilent Part Nr. 5182-3454) is
recommended.
4.2 PCI and NCI Conditions

For both PCI and NCI techniques,
the MSD software includes
sophisticated autotuning
programs. These autotune
programs assist adjusting the
flow of reagent gas and the
other parameters necessary for
successful CI operation.
These autotune values are a
good starting point. The value
for Electron Multiplier Voltage
(EM Voltage) normally needs to
be increased in over autotune
values by about 400 V. In PCI
mode, the results are primarily
dictated by choice and pressure
of the reagent gas, altering
autotune parameter values has
almost no benefits. In ammonia
PCI, increasing the pressure
typically increases the formation
of adduct ions. In NCI mode the
previously discussed tune
parameters are worth adjusting in
order to improve sensitivity. In
any case frequent tuning should
avoided, especially in ECNI mode
where the residual tuning gas can
increase the background for
several hours due to the extreme
sensitivity in ECNI. The analyte
amount (absolute amount onto
the column) recommended for
method development in scan
mode are 10ng for PCI and 1ng
or less for NCI.
Care must be taken to insure that

all solvents used for derivatization
are free of water. The solution
containing the sample that is to
be derivatized is evaporated
(blown-down) with purified dry
nitrogen introduced into the vial
by means of a capillary steel tube
(1/16 o.d.) or glass pipet. The
tubes opening is positioned some
millimeters above the liquid
surface and a gentle gas flow
(checked prior to placing over the
reaction vial) which forms only a
small depression on the surface,
is applied until the solvent has
completely evaporated. The
derivatization reagent is added to
the dry residue, the vial sealed,
and allowed to react for a specific
length of time, at a regulated and
usually elevated temperature.
Depending on the chemical nature
of the derivatizing reagent
(i.e., capable of degrading the
capillary column phase), it may
have to be removed by nitrogen
blow-down as described above and
reconstituted in an appropriate
solvent before the sample is
injected. Sometimes an adequate
dilution must be prepared.
According to experience, most
derivatives are unstable, even if
they are stored at low temperature.
In most cases, when analyzing
a sample instead of a standard,
derivatization happens to analytes
and to any reactive matrix
compounds present. Such
byproducts can complicate the
chromatogram and spectral
analysis and should be minimized
or eliminated by further sample
preparation steps or improved
chromatography.
4.3 Derivatization
Polar, chromatographically
difficult compounds frequently
need derivatization before analysis.
In NCI mode, derivatized analytes
show increased sensitivity. The
following recommendations refer
Chemically aggressive reagents

will deteriorate the stationary
phase of the column, especially
when applying splitless or
on-column injection. With split
injection, care should be taken to
avoid corrosion of the gas tubing
connected to the injector and that

the the gas regulation module
(EPC module) is not damaged.
5. Instrumentation
The documented applications
were executed with the Agilent
Technologies GC/MSD System:
Gas Chromatograph 6890plus,
split/splitless and On Column
Injector, Autosampler 7673
Mass Spectrometer MSD 5973N,
CI Option
HP Kayak XA, ChemStation
Software Vers. G1701CA
As reagent gas or buffer gas
methane (4.5) and ammonia
(3.5 or 4.0), Linde Gas AG, were
used. All gas supply tubes
were of stainless-steel material
and a gas purifier (Agilent Part
Nr. 1999-80410 used for only
methane) were installed between
the MSD and the gas bottles.
For ammonia, an appropriate,
corrosion resistant gas regulator
was used. The ammonia gas
stainless steel supply tube was
coiled and the pressure was
adjusted to approximately 7 psi
(0,5 bar) in order to avoid
generating droplets.
(For more information on
operating in ammonia, refer
to Agilent Technolgies Technique
Brief Nr. 5968-7844E).
6. Literatur
Chemical Ionization Mass
Spectrometry, 2nd Edition,
Alex G. Harrison, CRC Press,
ISBN 08493-4254-6
Introduction to Mass
Spectrometry, Chapter Six, 2,
J. Throck Watson, Raven Press,
New York
High Pressure Electron Capture
Mass Spectrometry,
W. B. Knighton, L. J. Sears and
E. P. Grimsrud,
Mass Spectrometry Reviews,
1996, 14, 327-343
Handbook of Analytical
Derivatization Reactions,
D. R. Knapp, John Wiley & Sons,
ISBN 0-471-03469-X
Handbook of Derivatives for
Chromatography, 2nd Edition,
K. Blau and J. Halket,
John Wiley & Sons,
ISBN 0-471-92699-X
Silylating Agents, Fluka,
ISBN 3-905617-08-0
Contents
Component
Summary
Acepromazine . . . . . . . . . . . . . . . 7
Alprazolam. . . . . . . . . . . . . . . . . 19
Amobarbital . . . . . . . . . . . . . . . . . 9
Barbital . . . . . . . . . . . . . . . . . . . . 9
Benzoylecgonine . . . . . . . . . . . . 25
Bromazepam . . . . . . . . . . . . . . . 19
Butethal . . . . . . . . . . . . . . . . . . . . 9
Chloramphenicol. . . . . . . . . . . . 29
Chlorphenoxamine . . . . . . . . . . 33
Chlorprothixene . . . . . . . . . . . . 35
Cholesterol. . . . . . . . . . . . . . . . . 95
Cimaterol . . . . . . . . . . . . . . . . . . 37
Clenbuterol . . . . . . . . . . . . . . . . 41
Cocaine . . . . . . . . . . . . . . . . . . . 47
Codeine . . . . . . . . . . . . . . . . . . . 49
Diazepam . . . . . . . . . . . . . . . . . . 19
Dimethindene . . . . . . . . . . . . . . 53
Dimetridazole . . . . . . . . . . . . . . 79
Diphenhydramine . . . . . . . . . . . 55
Estradiol . . . . . . . . . . . . . . . . . . 95
Estrone. . . . . . . . . . . . . . . . . . . . 95
Flunitrazepam . . . . . . . . . . . . . . 19
Lidocaine . . . . . . . . . . . . . . . . . . 57
Mabuterol . . . . . . . . . . . . . . . . . 59
MDA . . . . . . . . . . . . . . . . . . . . . . 63
(Methylendioxyamphetamine)
Mepivacaine . . . . . . . . . . . . . . . . 67
Methadone . . . . . . . . . . . . . . . . . 69
Metronidazole . . . . . . . . . . . . . . 79
Morphine . . . . . . . . . . . . . . . . . . 71
Nalorphine . . . . . . . . . . . . . . . . . 75
Orphenadrine. . . . . . . . . . . . . . . 83
Pentobarbital . . . . . . . . . . . . . . . . 9
Phenylbutazone . . . . . . . . . . . . . 85
Promethazine. . . . . . . . . . . . . . . 87
Propionylpromazine . . . . . . . . . 89
(Combelen)
Ractopamine . . . . . . . . . . . . . . . 91
Ronidazole . . . . . . . . . . . . . . . . . 79
Secobarbital. . . . . . . . . . . . . . . . . 9
Testosterone . . . . . . . . . . . . . . . 95
Tetrahydrocannabinol . . . . . . . 101
(THC)
Tetrahydrocannabinol
Carboxylic Acid . . . . . . . . . . . 105
(THCCOOH)
Triazolam . . . . . . . . . . . . . . . . . . 19
Zearalenone . . . . . . . . . . . . . . . 109
Barbiturates . . . . . . . . . . . . . 9
Amobarbital
Barbital
Butethal
Pentobarbital
Secobarbital
Benzodiazepines . . . . . . . . . 19
Alprazolam
Bromazepam
Diazepam
Flunitrazepam
Triazolam
Nitroimidazoles . . . . . . . . . . 79
Dimetridazole
Metronidazole
Ronidazole
Steroides . . . . . . . . . . . . . . . 95
Cholesterol
Estradiol
Estrone
Testosterone
Acepromazine
CAS-Nr. 61-00-7
Molecular formula: C19H22N2OS
GC-Parameters
Column: HP-5ms
Agilent Part Nr. 19091S-433
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Flow: 0.7ml/min, 30cm/sec
Mode: Constant Flow
Injection: Split, 250C
Oven Temp. Program
120C (0.3 min) - 20C/min to
300C (4 min)
MS-Parameter
Mode: EI SCAN
Tune: Atune
Temperatures:
Source 230C, Quad 150C
EI Spectrum, Acepromazine: m/z 326; M+
Mode: PCI/CH4 SCAN

Reagent Gas: Methane
Flow (Setting): 1 ml/min (20)
Tune: PCI-Methane Autotune
Temperatures:
EM Voltage: Tune + 400V
Mode: PCI/NH3 SCAN
Reagent Gas: Ammonia
Flow (Setting): 1 ml/min (20)
Tune: PCI-Ammonia Tune File
Results
Analyte Retention Time: 11.60min
Analyte Concentration: 4ng/l
Signal/Noise Ratios for TIC
PCI/CH4 Scan: > 18/1
PCI/NH3 Scan: > 25/1
PCI/CH4 Spectrum, Acepromazine: m/z 327, 355, 367; [M+H]+, [M+C2H5]+, [M+C3H5]+
PCI/NH3 Spectrum, Acepromazine: m/z 327; [M+H]+
Barbiturates
Amobarbital CAS-Nr. 57-43-2
Molecular formula: C11H18N2O3
Barbital CAS-Nr. 57-44-3
Butethal CAS-Nr. 77-28-1
Pentobarbital CAS-Nr. 76-74-4
Secobarbital CAS-Nr. 76-73-3
GC-Parameter
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Injection: Pulsed splitless, 250C
Oven Temperature Program
60C (1min) 20C/min to 180C
10C/min to 300C
MS-Parameter
Mode: EI SCAN
Tune: Atune
Temperatures:
Mode: PCI/CH4 SCAN
Flow (Setting): 1ml/min (20)
Temperatures:
Mode: ECNI/CH4 SCAN/SIM
Buffer Gas: Methane
Tune: ECNI-Methane Tune File
Temperatures:
Remarks
Derivatization with Pentafluorobenzylbromide (PFBB)
To 100l of the Barbiturate
Standard (SIGMA D 3155),
EI-Spectrum, Amobarbital, underivatised, m/z 226; M+
EI-Spectrum, Amobarbital, PFBB-derivative, m/z 406; M+
concentration 20ng/l each, diluted in

ethylacetate, 10l of the derivatization
reagent and 10l of triethylamine are
added and the mixtures is incubated
for 60 min at 60C. The reaction leads
to the mono-derivatives. An aliquot of
the derivatized solution is used for
GC/MSD measurement.
Caution: Only diluted samples are
injected in order to avoid column
stationary phase deterioration.
Results
Underivatized Barbiturates can be
measured without problems. The
derivatization improves sensitivity
and also increases molecular mass
(+181amu) which is advantageous
especially in SIM mode. A drastic
improvement is noticed in ECNI
mode. The signal/noise ratio for
1pg/l analyte concentration in ECNI
mode exceeds 200:1. Sensitivity for
the derivatives in PCI with NH3 is
very low.
PCI/CH4-Spectrum, Amobarbital, underivatised: m/z 227, 255, 267; [M+H]+, [M+C2H5]+, [M+C3H5]+
PCI/CH4-Spectrum, Amobarbital, PFBB-derivative: m/z 407, 435, 447; [M+H]+, [M+C2H5]+, [M+C3H5]+
ECNI/CH4-Spectrum, Amobarbital, PFBB-derivative: m/z 405; [M-H]-
10
EI-Spectrum, Barbital, underivatised, m/z 184; M+
EI-Spectrum, Barbital, PFBB-derivative, m/z 364; M+
PCI/CH4-Spectrum, Barbital, underivatised: m/z 185, 213, 225; [M+H]+, [M+C2H5]+, [M+C3H5]+
11
PCI/CH4-Spectrum, Barbital, PFBB-derivative: m/z 365, 393, 405; [M+H]+, [M+C2H5]+, [M+C3H5]+
ECNI/CH4-Spectrum, Barbital, PFBB-derivative: m/z 363; [M-H]-
EI-Spectrum, Butethal, underivatised, m/z 212; M+
12
EI-Spectrum, Butethal, PFBB-derivative, m/z 392; M+
PCI/CH4-Spectrum, Butethal, underivatised: m/z 213, 241, 253; [M+H]+, [M+C2H5]+, [M+C3H5]+
PCI/CH4-Spectrum, Butethal, PFBB-derivative: m/z 393, 421, 433; [M+H]+, [M+C2H5]+, [M+C3H5]+
13
ECNI/CH4-Spectrum, Butethal, PFBB-derivative: m/z 391; [M-H]-
EI-Spectrum, Pentobarbital, underivatised, m/z 226; M+
EI-Spectrum, Pentobarbital, PFBB-derivative, m/z 406; M+
14
PCI/CH4-Spectrum, Pentobarbital, underivatised: m/z 227, 255, 267; [M+H]+, [M+C2H5]+, [M+C3H5]+
PCI/CH4-Spectrum, Pentobarbital, PFBB-derivative, m/z 407, 435, 447; [M+H]+, [M+C2H5]+, [M+C3H5]+
ECNI/CH4-Spectrum, Pentobarbital, PFBB-derivative: m/z 405; [M-H]-
15
EI-Spectrum, Secobarbital, underivatised, m/z 238; M+
EI-Spectrum, Secobarbital, PFBB-derivative, m/z 418; M+
PCI/CH4-Spectrum, Secobarbital, underivatised: m/z 239, 267, 279; [M+H]+, [M+C2H5]+, [M+C3H5]+
16
PCI/CH4-Spectrum, Secobarbital, PFBB-derivative: m/z 419, 447, 459; [M+H]+, [M+C2H5]+, [M+C3H5]+
ECNI/CH4-Spectrum, Secobarbital, PFBB-derivative: m/z 417; [M-H]-
ECNI SIM, 5 Barbiturates

Analytes
in elution
order
Barbital
Butethal
Amobarbital
Pentobarbital
Secobarbital
RT Nominal Relative
(min)
ions
Signal/Noise
(m/z)
Ratio
14.52
363
1
15.37
391
1.03
15.63
405
1.00
15.93
405
1.15
16.13
417
0.72
Table 1
ECNI SIM, 5 Barbiturates, 1 pg/l each, see Table 1
17
18
Benzodiazepines
Alprazolam CAS-Nr. 28981-97-7
Molecular formula: C17H13ClN4
Bromazepam CAS-Nr. 1812-30-2
Molecular formula: C14H10BrN3O
Diazepam CAS-Nr. 439-14-5
Molecular formula: C16H13ClN2O
Flunitrazepam CAS-Nr. 1622-62-4
Molecular formula: C16H12FN3O3
Triazolam CAS-Nr. 28911-01-5
Molecular formula: C17H12Cl2N4
GC-Parameters
Column: HP-5ms
Agilent Part Nr.19091S-433
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Injection:
On Column: 100C/40C
Oven Temp.Program
Scan: 100C (0.3min)
SIM: 40C (0.3min) 25C/min to 300C (6min)
Flunitrazepam
Scan/SIM: 100C (0.3min)
MS-Parameter
Mode: EI SCAN
Tune: Atune
Temperatures:
Buffer Gas: Methane
Temperatures:
Remarks
Injection
Active surfaces in the inlet
system may causes discrimination.
For these measurements, on-column
injection technique with a fused
silica needle syringe was applied
to allow a better comparison.
EI-Spectrum, Alprazolam: m/z 308; M+
ECNI-Spectrum, Alprazolam: m/z 308; M-
19
ECNI Parameters
The commonly applied parameters
for improving ECNI measurements
(like Emission Current, Flow, NH3
Buffer Gas) showed no positive
effects.
Results
Sensitivity in ECNI Scan mode was
measured using concentrations
in the range of 0.3ng/l to 1ng/l
and is in that range. SIM mode
measurements were made at about
1000 times lower; 3pg/l to 10pg/l.
Flunitrazepam showed the highest
relative response (at < 200fg/l).
References
Application of Electron Capture
Negative Chemical Ionization
for the detection of a Date Rape
Drug
A. Negrusz, Ch. Moore, H. Prest
Agilent Pub. Nr. 5968-4364E
EI-Spectrum, Bromazepam: m/z 315; M+
ECNI-Spectrum, Bromazepam: m/z 315; M-
EI-Spectrum, Diazepam: m/z 284; M+
20
ECNI-Spectrum, Diazepam: m/z 284; M-
EI-Spectrum, Flunitrazepam: m/z 313; M+
ECNI-Spectrum, Flunitrazepam: m/z 313; M-
21
EI-Spectrum, Triazolam: m/z 342; M+
ECNI-Spectrum, Triazolam: m/z 342; M-
22
ECNI SIM
ECNI SIM
ECNI SIM, Alprazolam
Analytes
Retention
Time
(min)
Ions
(m/z)
Alprazolam
Bromazepam
Diazepam
Flunitrazepam
Triazolam
11.49
9.60
8.77
9.49
12.32
308, 310
315, 317
284, 286
313
306, 308
Table: Sensitivity Benzodiazepines
Bromazepam
Diazepam
Flunitrazepam
Triazolam
23
Signal/Noise
Relative to
Diazepam
(TIC)
0.6
0.08
1
16
0.7
24
Benzoylecgonine
CAS-Nr. 519-09-5
Molecular formula: C16H19NO4
GC-Parameters
Column: HP-5ms
Agilent Part Nr.19091S-433
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp. Program
70C (1min) 25C/min to
300C (5min)
MS-Parameter
Mode: EI SCAN
Tune: Atune
Temperatures:
Mode: PCI/CH4 SCAN

Temperatures:
Mode: PCI/NH3 SCAN
Remarks
Gentle evaporation with nitrogen

is repeated and the residue
redissolved in ethyl acetate.
b) Procedure as described above.
After the first evaporation the
residue, 80l of the PFPA reagent
and 20l of hexafluoro-isopropanol
(FLUKA 52517) are added and the
reaction mixture is incubated for
30 min at 70C. Then evaporation,
dilution and GC/MSD analysis.
Derivatization
a) Trimethyl silylation TMS with
MSTFA (Reagent: Fluka 69479)
b) Reaction with Pentafluoropropionic Acid Anhydride (PFPA)
(Reagent: Fluka 77292)
a) The standard solution (SIGMA
B 8900), concentration 100ng/l,
diluted in ethyl acetate, was
evaporated with a gentle nitrogen
flow. To the residue, 50l reagent
is added and the reaction mixture
is incubated for 20min at 60C.
Results
Derivatization is recommended.
In PCI/NH3 Mode, TMS Derivative,
the degree of fragmentation is
related to sample concentration
for this analyte.
Sensitivity is directly related
to the derivatization and to the
applied reagent gas, see table. In
SIM mode, analyte concentration
of 1pg/l is measured with signal/
noise ratio of approximately 10/1.
EI-Spectrum, Benzoylecgonine, underivatised: m/z 289; M+
EI-Spectrum, Benzoylecgonine, TMS Derivative: m/z 361; M+
25
EI-Spectrum, Benzoylecgonine, PFPA Derivative, -O-CH-(CF3)2: m/z 439; M+
PCI/CH4-Spectrum, Benzoylecgonine, 50ng/l, TMS Derivative: m/z 362, 390, 402; [M+H]+ , [M+C2H5]+, [M+C3H5]+
PCI/CH4-Spectrum, Benzoylecgonine, 10ng/l, PFPA Derivative, -O-CH-(CF3)2 : m/z 440, 468, 480; [M+H]+, [M+C2H5]+, [M + C3H5]+
26
PCI/NH3-Spectrum, Benzoylecgonine, 10ng/l, TMS Derivative: m/z 362; [M + H]+
PCI/NH3-Spectrum, Benzoylecgonine, 50ng/l, TMS Derivative: m/z 362; [M+H]+
PCI/NH3-Spectrum, Benzoylecgonine, 10ng/l, PFPA Derivative, -O-CH-(CF3)2: m/z 440; [M+H]+
27
PCI/NH3 SIM Mode

Derivative
Ion Mode
TMS
TMS
PFPA
PFPA
PCI/CH4
PCI/NH3
PCI/CH4
PCI/NH3
Peak to
Peak
Signal:Noise
Ratio
> 10 : 1
> 35 :1
> 100 : 1
> 200 : 1
Table : Benzoylecgonine, Sensitivity

(S/N), Scan Acquisition, 10ng/l each
Benzoylecgonine, PFPA Derivative,

Retention Time: 8.84min
1pg/l, SIM Ions: 318, 440 m/z
28
Chloramphenicol
CAS-Nr. 56-75-7
Molecular Formula: C11H12Cl2N2O5
CAS-Nr. O,O-TMS Derivative:
21196-84-9
GC-Parameter
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Injection: Pulsed splitless
Oven Temp. Program
70C (1min) - 30C/min to 150C
15C/min to 300C
MS-Parameter
Mode: EI SCAN
Tune: Atune
Temperatures:
Mode: PCI/CH4 SCAN

Temperatures:
Mode: PCI/NH3 SCAN
Flow (Setting): 1.75ml/min (35)
Buffer Gas: Methane
Temperatures:
SIM Tune+ 600V
C 0378), concentration 20ng/l,

diluted in ethyl acetate, is gently
evaporated under dry nitrogen.
To the residue, 50l reagent is
added and the reaction mixture
is incubated for 2 min at 50C.
Evaporation to dryness is repeated
and the residue diluted in hexane.
The solution is ready for injection.
Remarks
The greatest sensitivity is

achieved in ECNI SIM mode
which allows detection at 0.1 pg/l.
Calibration curves show good
linearity over the concentration
range of 0.1pg/l to 100pg/l.
Derivatization
Trimethyl silylation with
HMDS/TMCS at 2/1 in Pyridine
The standard solution (SIGMA
EI Spectrum, Chloroamphenicol, O,O-TMS Derivative: m/z 466; M+
PCI/CH4 Spectrum, Chloroamphenicol, O,O-TMS Derivative: m/z 467, 495, 507; [M+H]+, [M+C2H5]+, [M+C3H5]+
29
Results
Analyzing this compound as
O,O TMS derivative is preferred
over the parent.
PCI Scan sensitivity is on the
order of 20ng/l with methane
however ammonia produces
5 times greater sensitivity.
PCI/NH3 Spectrum, Chloroamphenicol, O,O-TMS Derivative: m/z 467, 484; [M+H]+ , [M+NH4]+
ECNI/CH4 Spectrum, Chloroamphenicol, O,O-TMS Derivative: m/z 466; M-
SIM Mode (Ions: 465.9, 467.9, 469.9 m/z)
ECNI/CH4 Chloroamphenicol, O,O-TMS Derivative, 1pg/l
ECNI/CH4 Chloroamphenicol, O,O-TMS Derivative, 0.1pg/l
30
Calibration Curves: ECNI/CH4 Chloroamphenicol, O,O-TMS Derivative

left: 0.1pg/l to 100pg/l, right: 0.1pg/l to 1pg/l
31
32
Chlorphenoxamine
CAS-Nr. 77-38-3
Molecular Formula: C18H22ClNO
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp Program
120C (0.3min) 20C/min to
300C (4min)
MS Parameters
Mode: EI - SCAN
Tune: Atune
Temperatures:
EI-Spectrum, Chlorphenoxamine: m/z 303; M+
Mode: PCI/CH4 SCAN

Temperatures:
Mode: PCI/NH3 SCAN
Results
PCI/NH3 Scan: >100/1
PCI/CH4-Spectrum, Chlorphenoxamine: m/z 304, 332, 344; [M+H]+ , [M+C2H5]+, [M+C3H5]+
PCI/NH3-Spectrum, Chlorphenoxamine: m/z 304; [M+H]+
33
34
Chlorprothixene
CAS-Nr. 113-59-7
Molecular Formula: C18H18ClNS
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp Program
300C (4min)
MS Parameters
Mode: EI SCAN
Tune: Atune
Temperatures:
EI-Spectrum, Chlorprothixene: m/z 315; M+
Mode: PCI/CH4 SCAN

Temperatures:
Mode: PCI/NH3 SCAN
Results
PCI/CH4-Spectrum, Chlorprothixene: m/z 316, 344, 356; [M+H]+, [M+C2H5]+ , [M+C3H5]+
PCI/NH3-Spectrum, Chlorprothixene: m/z 316 : [M+H]+
35
36
Cimaterol
CAS-Nr. 54239-37-1
Molecular Formula: C12H17N3O
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp. Program
280C (5min)
MS Parameters
Mode: EI SCAN
Tune: Atune
Temperatures:
Mode: PCI/CH4 SCAN
Temperatures:
Mode: PCI/NH3 SCAN

Buffer Gas: Methane
Temperatures:
EM Voltages: Tune + 400V
Remarks
Derivatizations: TMS and PPFA
a) Trimethyl silylation (TMS)
with BSTFA/TMCS
The standard solution (Boehringer
Ingelheim), concentration 1mg/ml,
diluted in methanol, is evaporated
to dryness with a gentle nitrogen
flow. To the residue, 50l reagent
and 125 l pyridine is added and
the reaction mixture is incubated
for 30min at 60C. Evaporation
is repeated and the residue is
redissolved in chloroform. The
solution is ready for injection.
EI-Spectrum, Cimaterol, underivatised: m/z 219; M+
37

The standard is treated as in a)
above. After the first evporation
the residue is treated with 80l
of the PFPA reagent, 20l of
Hexafluoroisopropanol (Fluka
52517) is added and the reaction
mixture is incubated for 30min at
70C, followed by evaporation,
dilution and GC/MSD analysis.
Results
The EI spectra of underivatised
and derivatized analytes show low
intensity for the molecular ion.
Both the TMS and PFPA
derivatization reactions form
the di-derivatives. The TMS
derivative response is higher
comparing to the PFPA derivative.
PCI/NH3 response shows
increased sensitivity by factor 2.6.
Both modes present the
characteristic PCI adduct ion
information. Sensitivity for the
TMS derivative in PCI/NH3 SIM
mode results in a signal-to-noise
ratio of >20:1 at an analyte
concentration of 50pg/l.
EI-Spectrum, Cimaterol, TMS Derivative: m/z 363; M+
EI-Spectrum, Cimaterol, PFPA Derivative: m/z 511; M+
PCI/CH4-Spectrum, Cimaterol, underivatised: m/z 220, 248, 260; [M+H]+, [M+C2H5]+, [M+C3H5]+; m/z 202, 230, 242; [M-OH]+, [M+C2H5-OH2]+, [M+C3H5-OH2]+
38
PCI/CH4-Spectrum, Cimaterol, TMS Derivative: m/z 364, 392, 404; [M+H]+, [M+C2H5]+, [M+C3H5]+
PCI/NH3-Spectrum, Cimaterol, TMS Derivative: m/z 364 [M+H]+
PCI/CH4-Spectrum, Cimaterol, PFPA Derivative: m/z 494, 522, 534; [M-OH]+, [M+C2H5-OH2]+, [M+C3H5-OH2]+
39
PCI/NH3-Spectrum, Cimaterol, PFPA Derivative: m/z 529; [M+NH4]+
ECNI/CH4-Spectrum, Cimaterol, PFPA Derivative: m/z 491; [M-HF]-
PCI/NH3 SIM Mode
Cimaterol, TMS Derivative, 50pg, Retention Time: 8.98min

Ions: 274, 364 m/z ; Signal/Noise: > 20/1
40
Clenbuterol
CAS-Nr. 37148-27-9
Molecular Formula: C12H18Cl2N2O
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Injection: Split & Pulsed splitless,
250C
Oven Temp. Program
Split: 100C (0.3min)
25C/min to 280C
Pulsed splitless: 80C (1min)
25C/min to 265C (1min)
MS Parameters
Mode: EI SCAN
Tune: Atune
Temperatures:
Mode: PCI/CH4 SCAN/SIM

Temperatures:
Mode: PCI/NH3 SCAN/SIM
Buffer Gas: Methane
Temperatures:
di-derivatives at a ratio of 1:0.75.

C 5423), concentration 1mg/ml,
in methanol, is evaporated with a
gentle nitrogen flow. To the residue
a mixture of Pyridine/TMS-Reagent
(2.5/1) is added and incubation
is done for 30min. The excess
reagent is evaporated and the
residue redissolved in chloroform.
The solution is ready for injection.
Remarks
Results
The degree of fragmentation is
related to the sample concentration
for this analyte. The response in
PCI/NH3 mode is higher comparing
to PCI/CH4 mode. Weak response
is observed in ECNI/CH4 mode.
Derivatization
Trimethyl silylation with BSTFA/
TMCS (Reagent: Fluka 15238)
Derivatization conditions (e.g,
incubation temperature and time)
have some impact on the product
formation. Applying 60C for
30min generates both mono- and
References
Analysis of Clenbuterol by
GC-MS, F. David, RIC,
Clenbuterol and Norandrosterone, B. Wst,
EI-Spectrum, Clenbuterol, underivatised: m/z 276; M+
EI-Spectrum, Clenbuterol, BSTFA-mono-derivative: m/z 348; M+
41
EI-Spectrum, Clenbuterol, BSTFA-di-derivative:, m/z 420; M+
PCI/CH4-Spectrum, Clenbuterol, underivatised: m/z 277, 305, 317; [M+H]+, [M+C2H5]+, [M+C3H5]+
PCI/CH4-Spectrum, Clenbuterol, BSTFA-mono-derivative, approx. 30ng: m/z 349 , 377 , 389 ; [M+H]+ , [M+C2H5]+ , [M+C3H5]+
42
PCI/CH4-Spectrum, Clenbuterol, BSTFA-mono-derivative, approx. 1ng, m/z 349, 377, 389; [M+H]+, [M+C2H5]+, [M+C3H5]+
PCI/CH4-Spectrum, Clenbuterol, BSTFA-di-derivative, approx. 40ng: m/z 421, 449, 461; [M+H]+, [M+C2H5]+, [M+C3H5]+
PCI/CH4-Spectrum, Clenbuterol, BSTFA-di-derivative, approx. 1ng: m/z 421, 449, 461; [M+H]+, [M+C2H5]+, [M+C3H5]+
43
PCI/NH3-Spectrum, Clenbuterol, underivatised: m/z 277; [M+H]+
PCI/NH3-Spectrum, Clenbuterol, mono-derivative, approx. 30ng: m/z 349; [M+H]+
PCI/NH3-Spectrum, Clenbuterol, mono-derivative, approx. 1ng: m/z 349; [M+H]+
44
PCI/NH3-Spectrum, Clenbuterol, approx. 40ng, di-derivative: m/z 421; [M+H]+
PCI/NH3-Spectrum, Clenbuterol, di-derivative, approx. 1ng: m/z 421; [M+H]+
ECNI/CH4-Spectrum, Clenbuterol, approx. 1ng, underivatised: m/z 276; M-
45
ECNI/CH4-Spectrum, Clenbuterol, approx. 1ng, di-derivative: m/z 420; M-
SIM Mode
PCI/CH4, Clenbuterol, approx. 15pg, mono & di-derivative: S/N 70/1 & 5/1
PCI/NH3, Clenbuterol, approx. 1.5pg, mono &di-derivative: S/N 55/1 & 8/1
ECNI/CH4, Clenbuterol, approx. 15 pg, di-derivative: S/N 30/1
46
Cocaine
CAS-Nr. 50-36-2
Molecular Formula: C17H21NO4
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp.Program
300C (5min)
MS Parameters
Mode: EI SCAN
Tune: Atune
Temperatures:
Mode: PCI/CH4 SCAN
Temperatures:
Mode: PCI/NH3 SCAN
EI-Spectrum, Cocaine: m/z 303: M+
PCI/CH4-Spectrum, Cocaine: m/z 304, 332, 344; [M+H]+ , [M+C2H5]+ , [M+C3H5]+
47
Results
In PCI mode, SCAN acquisition,
ammonia is the preferred reagent
gas due to higher response
compared to methane. In SIM
mode there were no significant
differences in responses
observed. In PCI/CH4 SIM mode
signal/noise ration of >20/1 was
measured for 10pg/l. ECNI
showed no relevant spectrum.
PCI/NH3-Spectrum, Cocaine: m/z 304; [M+H]+
PCI/CH4 SIM Mode
Cocaine, Retention Time: 9.50min, 10pg/l: Ions 182, 304 m/z ;

S/N > 25/1
48
Codeine
CAS-Nr. 76-57-3
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp. Program
(5min)
MS Parameters
Mode: EI SCAN
Tune: Atune
Temperatures:
Mode: PCI/CH4 SCAN

Temperatures:
Mode: PCI/NH3 SCAN
Buffer Gas: Methane
Temperatures:
Remarks
Derivatization
Reaction with Pentafluoropropionic Acid Anhydride (PFPA)
EI-Spectrum, Codeine, underivatised: m/z 299 ; M+
EI-Spectrum, Codeine, PFPA Derivative: m/z 445 ; M+
49
C 1653), concentration 100ng/l in

ethyl acetate, is evaporated with a
gentle flow of nitrogen. To the
residue 80l of the PFPA reagent
and 20l of Hexafluoroisopropanol
(Fluka 52517) is added and the
30min at 70C. Evaporation is
repeated and the residue redissolved
in ethyl acetate. The solution is
ready for injection and analysis.
Results
Even the underivatized analyte is
measured without chromatographic
discrimination. In PCI mode, the
response of the derivatized analyte
was 2-times higher using ammonia
reagent gas than methane.
ECNI/CH4 SIM measurements were
highly sensitive for the derivatized
analyte; 5pg/l resulted in
signal/noise ratio of >125/1.
The acetylated analyte showed no
significant ECNI spectrum.
PCI/CH4-Spectrum, Codeine, PFPA Derivative: m/z 446, 474, 486; [M+H]+, [M+C2H5]+, [M+C3H5]+
PCI/NH3-Spectrum, Codeine, PFPA Derivative: m/z 446, 463; [M+H]+, [M+NH4]+
ECNI/CH4-Spectrum, Codeine, PFPA Derivative: m/z 425; [M-HF]-
50
ECNI/CH4 SIM
Codeine, PFPA Derivative, 5pg, Retention Time: 10.08min

Ion: 425 m/z, Signal/Noise 150/1
51
52
Dimethindene
CAS-Nr. 5636-83-9
Molecular Formula: C20H24N2
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp. Program
300C (4min)
MS Parameters
Mode: EI SCAN
Tune: Atune
Temperatures:
EI-Spectrum, Dimethindene: m/z 292; M+
Mode: PCI/CH4 SCAN

Temperatures:
Mode: PCI/NH3 SCAN
Results
PCI/CH4 Scan: > 5/1
PCI/CH4-Spectrum, Dimethindene: m/z 293, 321, 333; [M+H]+, [M+C2H5]+, [M+C3H5]+
PCI/NH3-Spectrum, Dimethindene: m/z 293; [M+H]+
53
54
Diphenhydramine
CAS-Nr. 58-73-1
Molecular Formula: C17H21NO
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp. Program
300C (4min)
MS Parameters
Mode: EI SCAN
Tune: Atune
Temperatures:
EI-Spectrum, Diphenhydramine: m/z 255; M+
Mode: PCI/CH4 SCAN

Temperatures:
Mode: PCI/NH3 SCAN
Results
PCI/CH4 Scan: >500/1
PCI/NH3 Scan: >80/1
PCI/CH4-Spectrum, Diphenhydramine: m/z 256, 284, 296; [M+H]+ , [M+C2H5]+, [M+C3H5]+
PCI/NH3-Spectrum, Diphenhydramine: m/z 256; [M+H]+
55
56
Lidocaine
CAS-Nr. 137-58-6
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp. Program
300C (4min)
MS Parameters
Mode: EI SCAN
Tune: Atune
Temperatures:
EI-Spectrum, Lidocaine: m/z 234; M+
Mode: PCI/CH4 SCAN

Temperature:
Source 250C , Quad 106C
Mode: PCI/NH3 SCAN
Results
PCI/CH4 Scan: >350/1
PCI/NH3 Scan: >80/1
PCI/CH4-Spectrum, Lidocaine: m/z 235, 263, 275; [M+H]+, [M+C2H5]+, [M+C3H5]+
PCI/NH3-Spectrum, Lidocaine: m/z 235; [M+H]+
57
58
Mabuterol
CAS-Nr. 56341-08-3
Molecular Formula: C13H18ClF3N2O
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp. Program
70C (1min) 25C/min
to 280C (5min)
MS Parameters
Mode: EI SCAN
Tune: Atune
Temperatures:
Mode: PCI/CH4 SCAN
Temperatures:

Buffer Gas: Methane
Temperatures:
Remarks
Derivatization
a) Trimethyl silylation with
BSTFA/TMCS
b) Reaction with Pentafluoropropionic Acid Anhydride PFPA
a) 100l of the hydrochloride
standard (Boehringer Ingelheim),
concentration 1.6mg/ml, is
dissolved in methanol and
evaporated with a gentle flow of
nitrogen. To the residue, 50l of
derivatization reagent and 125l
of dry pyridine are added and the
30min at 60C. Gentle evaporation
with nitrogen is repeated and the
residue dissolved in chloroform.
EI-Spectrum, Mabuterol, underivatised, m/z 310; M+
59

After the first evaporation to the
residue, 80l of the derivatization
reagent and 20l of hexafluoro
isopropanol (Fluka 52517) are
Then evaporation, dilution and
GC/MSD analysis.
Results
The EI spectra of the underivatized
and derivatized analytes show low
intensity for the molecular ion.
The TMS derivatization react to
form a mono-derivative and the
PFPA derivatization forms a
di-derivative. The PFPA spectra
show M-18 fragmentation. In
PCI/CH4 mode the derivatives
generate the molecular ions and
show the characteristic adduct
ions. The measurements of the
TMS derivatives in PCI/NH3 Scan
mode are by factor 6 more sensitive
than the PCI/CH4 measurements.
The PFPA derivative shows less
response than the TMS derivative
in PCI mode.
An improvement is noticed in ECNI
mode for the PFPA derivative.
In SIM mode, the signal/noise ratio
is approximately 40:1 for an analyte
concentration of 200fg/l.
EI-Spectrum, Mabuterol, TMS mono-derivative, m/z 382; M+
EI-Spectrum, Mabuterol, PFPA di-derivative, m/z 602; M+
PCI/CH4-Spectrum, Mabuterol, underivatised, m/z 311, 339, 351; [M + H]+ , [M + C2H5 ]+, [M + C3H5 ]+
60
PCI/CH4-Spectrum, Mabuterol, TMS mono-derivative, m/z 383, 411, 423; [M + H]+, [M + C2H5 ]+, [M + C3H5 ]+
PCI/NH3-Spectrum, Mabuterol, TMS mono-derivative, m/z 383; [M + H]+
PCI/CH4-Spectrum, Mabuterol, PFPA di-derivative, m/z 585, 613, 625; [M-18 + H]+, [M-18 + C2H5 ]+, [M-18 + C3H5 ]+
61
PCI/NH3-Spectrum, Mabuterol, PFPA di-derivative, m/z 602; M +
ECNI/CH4-Spectrum, Mabuterol, PFPA di-derivative, molecular mass = 602 u
ECNI/CH4 SIM Mode
Mabuterol, PFPA derivative, 200fg,

Ions: m/z 507/509, Signal/Noise: 40/1
62
MDA
3,4 Methylendioxyamphetamine
CAS-Nr. 4764-17-4
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp. Program
(5min)

Temperatures:
Mode: PCI/NH3 SCAN
Mode:
ECNI/CH4/NH3 SCAN/SIM
Buffer Gas: Methane
Buffer Gas: Ammonia
Temperatures:
Remarks
MS Parameters
Derivatization
a) Trifluoroacetylation (TFA) with
MBTFA (Reagent: Fluka 65943)
b) Reaction with Pentafluoropropionic Acid Anhydride PFPA
Mode: EI SCAN
Tune: Atune
Temperatures:
Mode: PCI/CH4 SCAN
a) 100l of the standard

(SIGMA M 3272), 100ng/l MDA
concentration, is dissolved in
ethyl acetate and evaporated with
a gentle flow of nitrogen. To the
residue 50l derivatization
EI-Spectrum, MDA, TFA derivative, m/z 275; M+
63
reagent is added and the solution

dissolved in ethyl acetate.
reagent and 20l of hexafluoroisopropanol (Fluka 52517) are
GC/MSD analysis.
Results
Derivatization is recommended
for this analyte. In PCI/NH3 mode
the analyte spectrum shows the
NH4 adduct ion as base peak.
Signal is a factor 1.5 more
sensitive compared to PCI with
CH4. In ECNI mode the TFA
derivative response is relatively
low. PFPA derivatization is the
reaction of choice for both PCI
and ECNI modes. Fragmentation
and sensitivity are related to the
choice of derivatizing reagent and
buffer gas. In ECNI/NH3 SIM
mode the signal:noise ratio is
approximately 65:1 for the 5pg
PFPA derivative.
PCI/CH4-Spectrum, MDA, TFA derivative, m/z 276, 304, 316; [M +H]+, [M + C2H5 ]+, [M + C3H5 ]+
PCI/NH3-Spectrum, MDA, TFA derivative, m/z 293; [M + NH4 ]+
EI-Spectrum, MDA, PFPA derivative, m/z 325; M+
64
PCI/CH4-Spectrum, MDA, PFPA derivative, m/z 326, 354, 366; [M +H]+, [M + C2H5 ]+, [M + C3H5 ]+
PCI/NH3-Spectrum, MDA, PFPA derivative, m/z 343; [M + NH4 ]+
ECNI/CH4-Spectrum, MDA, PFPA derivative, m/z 305, 324; [M-HF]-, [M-H]-
65
ECNI/NH3-Spectrum, MDA, PFPA derivative, m/z 305, 324; [M-HF]-, [M-H]-
ECNI/NH3 SIM Mode
MDA, PFPA derivative, 5pg,

Ions: m/z 305, 324; Signal: Noise 65/1
66
Mepivacaine
(Carbocaine)
CAS-Nr. 96-88-8
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp. Program
300C (4min)
MS Parameters
Mode: EI SCAN
Tune: Atune
Temperatures:
EI-Spectrum, Mepivacaine: m/z 246; M+
Mode: PCI/CH4 SCAN

Temperature:
Mode: PCI/NH3 SCAN
Results
PCI/CH4-Spectrum, Mepivacaine: m/z 247, 275, 287; [M+H]+, [M+C2H5]+, [M+C3H5]+
PCI/NH3-Spectrum, Mepivacaine: m/z 247; [M+H]+
67
68
Methadone
CAS-Nr. 76-99-3
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp. Program
300C (4min)
MS Parameters
Mode: EI SCAN
Tune: Atune
Temperatures:
EI-Spectrum, Methadone: m/z 309; M+
Mode: PCI/CH4 SCAN

Temperatures:
Mode: PCI/NH3 SCAN
Results
PCI/CH4-Spectrum, Methadone: m/z 310, 338, 350; [M+H]+, [M+C2H5]+, [M+C3H5]+
PCI/NH3-Spectrum, Methadone: m/z 310; [M+H]+
69
70
Morphine
CAS-Nr. 57-27-2
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp. Program
(5min)
MS Parameters
Mode: EI SCAN
Tune: Atune
Temperatures:
Mode: PCI/CH4 SCAN
Temperatures:
Mode: PCI/NH3 SCAN
Buffer Gas: Methane
Temperatures:
Remarks
Derivatization
Reaction with Pentafluoropropionic Acid Anhydride (PFPA)
100l of the standard (SIGMA
M 9524), concentration 100ng/l,
dissolved in ethyl acetate is
EI-Spectrum, Morphine, underivatized, m/z 285; M+
EI-Spectrum, Morphine, Trifluoroacetyl derivative, m/z 477; M+
71

nitrogen. To the residue 80l
hexafluoroisopropanol are added
and the mixture is incubated for
residue redissolved in ethyl
acetate. The solution is ready
GC/MSD analysis.
Results
The trifluoroacetylation (TFA)
with MBTFA leads in EI mode to
a moderately intense molecular
ion. In ECNI mode the TFA analyte
spectrum shows no distinctive
results for the derivative.
The PFPA derivatization is suitable
for both PCI/NH3 and for ECNI/CH4
measurements. The signal-to-noise
ratio for 1pg analyte PFPA
derivative in ECNI/CH4 mode
is approximately 30:1.
EI-Spectrum, Morphine, PFPA derivative, m/z 577; M+
PCI/CH4-Spectrum, Morphine, PFPA derivative, m/z 578, 606, 618; [M + H]+, [M + C2H5 ]+, [M + C3H5]+
PCI/NH3-Spectrum, Morphine, PFPA derivative, m/z 578; [M + H]+
72
ECNI/CH4-Spectrum, Morphine, PFPA derivative, m/z 537, 557, 577; [M 2(HF)]-, [M-HF]-, [M]-
ECNI/CH4 SIM Mode

Acquisition
Mode
Analyte
conc.
EI-Scan
PCI/CH4-Scan
PCI/NH3-Scan
ECNI/CH4-Scan
ECNI/CH4-SIM
10ng/l
10ng/l
10ng/l
10ng/l
1pg/l
Approximate
Signal/Noise
Ratio
250/1
70/1
150/1
110/1
30/1
Table: Morphine, PFPA derivative, Sensitivity (S/N), EI/PCI/ECNI, Scan/SIM
Morphine, PFPA derivative, Retention Time: 9.89min

1pg/l, Ions: m/z 537, 557; S/N 30/1
73
74
Nalorphine
CAS-Nr. 62-67-9
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp. Program
(5min)
MS Parameters
Mode: EI SCAN
Tune: Atune
Temperatures:
Mode: PCI/CH4 SCAN
Temperatures:
Mode: PCI/NH3 SCAN
Buffer Gas: Methane
Temperatures:
Remarks
Derivatization
Reaction with Pentafluoropropionic
Acid Anhydride (PFPA)
100l of the standard (SIGMA
N 0762), concentration 100ng/l,
dissolved in ethyl acetate is
EI-Spectrum, Nalorphine, underivatized, m/z 311; M+
EI-Spectrum, Nalorphine, Trifluoroacetyl derivative, m/z 503; M+
75

nitrogen. To the residue, 80l of
hexafluoroisopropanol are added
and the mixture is incubated for
residue redissolved in ethyl
acetate. The solution is ready
GC/MSD analysis.
Results
The trifluoroacetylation (TFA)
with MBTFA leads in EI mode to
a moderately intense molecular
ion. In ECNI mode the TFA
analyte spectrum exhibits no
distinctive features.
The PFPA derivatization is
suitable for PCI/NH3 and for
ECNI/CH4 measurements. The
signal/noise ratio for 1pg analyte
PFPA derivative in ECNI/CH4
mode is approximately 15:1.
EI-Spectrum, Nalorphine, PFPA derivative, m/z 603; M+
PCI/CH4-Spectrum, Nalorphine, PFPA derivative, m/z 604, 632, 644; [M + H]+, [M + C2H5 ]+, [M + C3H5]+
PCI/NH3-Spectrum, Nalorphine, PFPA derivative, m/z 604; [M + H]+
76
ECNI/CH4-Spectrum, Nalorphine, PFPA derivative, m/z 563, 583, 603; [M 2(HF)]-, [M HF]-, M-
ECNI/CH4 SIM Mode

Acquisition
Mode
Analyte
conc.
EI-Scan
PCI/CH4-Scan
PCI/NH3-Scan
ECNI/CH4-Scan
ECNI/CH4-SIM
10ng/l
10ng/l
10ng/l
10ng/l
1pg/l
Approximate
Signal/Noise
Ratio
240/1
75/1
110/1
200/1
15/1
Table: Nalorphine, PFPA derivative, Sensitivity (S/N), EI/PCI/ECNI, Scan/SIM
Nalorphine, PFPA derivative, Retention Time: 10.30min

1pg/L, Ions: m/z 563, 583; S/N: 16/1
77
78
Nitroimidazoles
Dimetridazole
CAS-Nr. 551-92-8
Molecular Formula: C5H7N3O2
Ronidazole
CAS-Nr. 7681-76-7
Metronidazole
CAS-Nr. 443-48-1
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp. Program
MS Parameters
Mode: EI SCAN
Tune: Atune
Dimetridazole
Temperatures:
Buffer Gas: Methane
Buffer Gas: Ammonia
Temperatures:
EM Voltage Scan: Tune + 300V
EM Voltage SIM: Tune + 400V
of the reagent is added and

incubated at 15min at 60C.
The derivatized solutions are used
for GC/MSD measurements.
Derivatization
Ronidazole (Reagent: SIGMA
R 7635) and Metronidazole
(Reagent: SIGMA M 1547)
can be silylated with MSTFA
Each standard solution,
concentration 1ng/l in ethyl
acetate, is evaporated with a gentle
nitrogen flow. To the residue, 50l
Results
The spectrum of Ronidazole
shows no molecular ions either
in EI or in ECNI mode. Spectral
base peaks of the analytes are
related to the fragmentation at
the carbamide ester positions.
The degree of fragmentation is
affected by choice of the buffer
gas. Both methane and ammonia
show different responses.
Considering absolute signal
intensitiy, ammonia is the preferred
buffer gas. However the signal/noise
ratios indicate that both gases
perform comparably. The relative
S/N ratios in SIM mode for these
nitroimidazoles are approximately:
Dimetridazole : Metronidazol :
Ronidazole = 100 : 10 : 1.
Ronidazole
Metronidazole
Remarks
EI-Spectrum, Dimetridazole, m/z 141; M+
79
ECNI/CH4-Spectrum, Dimetridazole, m/z 141; M-
EI-Spectrum, Ronidazole, TMS derivative, molecular mass = 272 u
ECNI/CH4-Spectrum, Ronidazole, TMS derivative, molecular mass = 272 u
80
ECNI/NH3-Spectrum, Ronidazole, TMS derivative, molecular mass = 272 u
EI-Spectrum, Metronidazole, TMS derivative, m/z 243; M+
ECNI/CH4-Spectrum, Metronidazole, TMS derivative, m/z 243; M-
81
Scan Mode, ECNI, 1ng each

3
1
2
TIC of Dimetridazole(1), Ronidazole(2), Metronidazole(3)

Buffer Gas: Methane, bottom; Ammonia, top
SIM Mode, ECNI/CH4, 1 pg each
Ronidazole, S/N 6/1

Ions m/z 139/229
Metronidazole, S/N 60/1

Ion m/z 243
Dimetridazole, S/N 600/1

Ion m/z 141
82
Orphenadrine
CAS-Nr. 83-98-7
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp. Program
300C (4min)
MS Parameters
Mode: EI SCAN
Tune: Atune
Temperature:
EI-Spectrum, Orphenadrine: m/z 269; M+
Mode: PCI/CH4 SCAN

Temperatures:
Mode: PCI/NH3 SCAN
Results
PCI/CH4-Spectrum, Orphenadrine: m/z 270, 298, 310; [M+H]+ , [M+C2H5]+ , [M+C3H5]+
PCI/NH3-Spectrum, Orphenadrine: m/z 270; [M+H]+
83
84
Phenylbutazone
CAS-Nr. 50-33-9
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp. Program
(2min)
MS Parameters
Mode: EI SCAN
Tune: Atune
Temperatures:
EI-Spectrum, Phenylbutazone, m/z 308; M+

Buffer Gas: Methane
Temperatures:
Remarks
Results
In EI mode, the analyte shows a
moderately intense molecular ion.
ECNI/CH4 mode generates the
molecular anion and SIM
measurements in this mode result
in an approximate signal/noise
ratio of 15:1 for 1pg of analyte.
ECNI/CH4-Spectrum, Phenylbutazone, m/z 308; M-
ECNI/CH4 SIM Mode
Phenylbutazone, 1pg, Retention Time: 10.32min

Ion: m/z 308, Signal/Noise 7/1
85
86
Promethazine
CAS-Nr. 60-87-7
Molecular Formula: C17H20N2S
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp. Program
300C (4min)
MS Parameters
Mode: EI SCAN
Tune: Atune
Temperatures:
EI-Spectrum, Promethazine: m/z 284 ; M+
Mode: PCI/CH4 SCAN

Temperatures:
Mode: PCI/NH3 SCAN
Results
PCI/CH4-Spectrum, Promethazine: m/z 285, 313, 325; [M+H]+ , [M+C2H5]+ , [M+C3H5]+
PCI/NH3-Spectrum, Promethazine: m/z 285; [M+H]+
87
88
Propionylpromazine
Combelen
CAS-Nr. 3568-24-9
Molecular Formula: C20H24ON2S
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp. Program
300C (4min)
MS Parameters
Mode: EI SCAN
Tune: Atune
Temperatures:
EI-Spectrum, Propionylpromazine: m/z 340; M+
Mode: PCI/CH4 SCAN

Temperatures:
Mode: PCI/NH3 SCAN
Results
PCI/CH4 Scan: > 8/1
PCI/CH4-Spectrum, Propionylpromazine: m/z 327, 355, 367; [M+H-CH2]+, [M+C2H5-CH2]+, [M+C3H5-CH2]+
PCI/NH3-Spectrum, Propionylpromazine: m/z 341; [M+H]+
89
90
Ractopamine
CAS-Nr. 99095-19-9 (HCl)
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp. Program
(5min)
MS Parameters
Mode: EI SCAN
Tune: Atune
Temperatures:
Mode: PCI/CH4 SCAN

Temperatures:
Remarks
Derivatization
Silylation (TMS) with BSTFA/TMCS
100l of the hydrochloride standard
(Lilly Research Laboratories),
concentration 1.6mg/ml in methanol,
EI-Spectrum, Ractopamine, underivatized, molecular mass = 301 u
PCI/CH4-Spectrum, Ractopamine, underivatized, molecular mass = 301 u
91
is evaporated with a gentle flow

of nitrogen. To the residue, 50l
pyridine are added and the mixture
incubated for 30min at 60C. Gentle
evaporation with nitrogen is repeated
and the residue redissolved in
chloroform. The solution is ready
GC/MSD analysis.
Results
Derivatization is recommended due
to the polar nature of the analyte.
The TMS reaction leads to the triderivative. The underivatised analyte
shows no protonated molecule or
molecular adducts in PCI/CH4. The
PCI/NH3 reaction of the silylated
analyte is the method of choice.
In PCI/NH3 SIM the signal/noise
ratio for 100pg of analyte is
calculated as approximately 30/1.
EI-Spectrum, Ractopamine, TMS derivative, m/z 517; M+
PCI/CH4-Spectrum, Ractopamine, TMS derivative, m/z 518, 546, 558; [M + H]+, [M + C2H5 ]+, [M + C3H5 ]+
PCI/NH3-Spectrum, Ractopamine, TMS derivative, m/z 518, 535; [M + H]+, [M + NH4]+
92
PCI/NH3 SIM Mode
Ractopamine, TMS derivative, 100pg,

Ions: m/z 428, 518; Signal/Noise 30/1
93
94
Steroids
Cholesterol CAS-Nr. 57-88-5
Molecular Formula: C27H46O
Estradiol CAS-Nr. 7681-76-7
Molecular Formula: C18H24O2
Estrone CAS-Nr. 443-48-1
Testosterone CAS-Nr. 58-22-0
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp. Program
(5.5min)
MS Parameters
Mode: EI SCAN
Tune: Atune
Temperatures:

Buffer Gas: Methane
Temperatures:
EM Voltage Scan: Tune + 400V
EM Voltage SIM: Tune + 800V
Optimization of Tune Parameters
Emission Current: Tune + 150A
Remarks
Derivatization
The analytes (Cholesterol/Estradiol/
Estrone/Testosterone SIGMA
C 8667/E 8750/ E 9750/ T 1500) can
be derivatized with BSTFA and
with Pentafluorophenyl/DMCS as
well as other approaches.
Derivatization with TMS
To 200l of the analytes at a
concentration of 1ng/l each in
chloroform, 100l of the
derivatization reagent (BSTFA,
Fluka 15238) is added and the
mixture is incubated for 30min at
60C. After evaporation under a
gentle flow of nitrogen, the residue
EI-Spectrum, Cholesterol TMS derivative, m/z 458; M+
EI-Spectrum, Cholesterol PFPh derivative, m/z 610; M+
95
is redissolved in ethyl acetate; the

solution is ready for injection.
Derivatization with PFPh
The analytes, concentration 1-2mg/ml
each, are dissolved in 100l acetonitrile and 100l pyridine is added.
The mixture is shaken (VortexMinishaker) for 2min and to the
clear solution 50l of derivatization
reagent (Pentafluorophenyl/
DMCS, Fluka 76750) is added. Be
unconcerned with the precipitate.
The reaction takes 20min at room
temperature. After addition of
800l chloroform the precipitate
dissolves and an aliquot is diluted
prior to injection.
Caution: PFPh derivatives are
unstable even at low temperatures.
Results
The TMS derivatives show weak
response in ECNI Scan mode, e.g.
for Cholesterol, concentration
18ng/l, signal/noise ratio 10/1.
Favourable responses are obtained
with the PFPh derivatives of Estrone
and Estradiole: low concentrations
( 200fg/l) can be detected in
SIM mode.
ECNI/CH4-Spectrum, Cholesterol TMS derivative, m/z 458; M-
ECNI/CH4-Spectrum, Cholesterol, PFPh derivative, m/z 610; M-
EI-Spectrum, Estradiol TMS derivative, m/z 416; M+
96
EI-Spectrum, Estradiol PFPh derivative, m/z 720; M+
ECNI/CH4-Spectrum, Estradiol TMS derivative, m/z 416; M-
ECNI/CH4-Spectrum, Estradiol PFPh-derivative, m/z 720; M-
97
EI-Spectrum, Estrone TMS derivative, m/z 342; M+
EI-Spectrum, Estrone PFPh-derivative, m/z 494; M+
ECNI/CH4-Spectrum, Estrone TMS-derivative, M-: 342m/z
98
ECNI/CH4-Spectrum, Estrone PFPh derivative, m/z 494; M-
EI-Spectrum, Testosterone, TMS derivative, m/z 360; M+
EI-Spectrum, Testosterone, PFPh derivative, m/z 512; M+
99
ECNI/CH4-Spectrum, Testosterone, TMS derivative, m/z 360; M-
ECNI/CH4-Spectrum, Testosterone, PFPh derivative, molecular mass = 512 u
ECNI/CH4 SIM
Estrone (left); Estradiol (right);

SIM Ions: m/z 494; m/z 720; 0.2pg/l each
100
Tetrahydrocannabinol
(d9-THC) CAS-Nr. 1972-08-3
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp. Program
(5min)
MS Parameters
Mode: EI SCAN
Tune: Atune
Temperatures:
Mode: PCI/CH4 SCAN
Temperatures:
Mode: PCI/NH3 SCAN

Mode: ECNI/CH4 SCAN
Buffer Gas: Methane
Temperatures:
Emission Current: 150A
Remarks
Derivatization
a) Trifluoroacetylation (TFA) with
MBTFA (Reagent: Fluka 65943)
a) 100l of the standard (SIGMA
T 4764) at a concentration of
100ng/l is dissolved in ethyl
acetate and evaporated with a
residue 50l derivatization
After the first evaporation of the
EI-Spectrum, Tetrahydrocannabinol, underivatized, m/z 314; M+
101

GC/MSD analysis.
Results
Even the underivatized analyte at
a concentration 10ng/l can be
measured without chromatographic
discrimination. The described
derivatization reactions lead to
THC by-products, not considered
here. The trifluoroacetylated
analyte can be measured in ECNI
mode, however the PFPA derivative
shows no significant ECNI
spectrum. The cited literature
give additional information about
THC determinations in PCI and
ECNI modes.
Literature
Cannabinoids in Blood:
Advantage of Positive Chemical
Ionization in Mass Spectroscopy
Harry Prest,
Agilent Pub. Nr. 5967-6103
Determining Cannabinoids in
Blood Using Electron Capture
Negative Chemical Ionization
Harry Prest,
Agilent Pub. Nr. 5967-6331
EI-Spectrum, Tetrahydrocannabinol, TFA derivative, m/z 410; M+
EI-Spectrum, Tetrahydrocannabinol, PFPA derivative, m/z 460; M+
PCI/CH4-Spectrum, Tetrahydrocannabinol, PFPA derivative, m/z 461, 489, 501; [M + H]+, [M + C2H5]+, [M + C3H5 ]+
102
PCI/NH3-Spectrum, Tetrahydrocannabinol, PFPA derivative, m/z 461, 478; [M + H]+, [M + NH4]+
ECNI/CH4-Spectrum, Tetrahydrocannabinol, TFA derivative, m/z 410; M-
103
104
THC COOH
11-Nor-d9-Tetrahydrocannabinol
-9-Carboxylic Acid
CAS-Nr. 56354-06-4
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp. Program
(3min)
MS Parameters
Mode: EI SCAN
Tune: Atune
Temperatures:
Mode: PCI/CH4 SCAN
Temperatures:
Mode: PCI/NH3 SCAN
Mode: ECNI/CH4/NH3 SCAN/SIM
Buffer Gas: Methane
Buffer Gas: Ammonia
Temperatures:
Emission Current: 150A
Remarks
Derivatization
a) Silylation (TMS) with BSTFA/
N 3142) at a concentration of
100ng/l is dissolved in ethyl
acetate and evaporated with a
residue, 50l of derivatization
EI-Spectrum, Tetrahydrocannabinol Carboxcylic Acid, TMS derivative, m/z 488; M+
105

After the first evaporation, 80l
of the derivatization reagent and
20l of hexafluoroisopropanol
(Fluka 52517) are added to the
residue and the reaction mixture
GC/MSD analysis.
Results
Derivatization is recommended
for this polar analyte. The analyte
response is related to the choice
of reagent/buffer gases applied.
The PFPA derivative is more
suitable for ECNI measurements
and the highest sensitivity is
achieved in ECNI/NH3 SIM mode.
Literature
Zum Nachweis von 11-nor-d9Tetrahydrocannabinolcarbonsure
in menschlichen Haaren mittels
GC/MS
U. Dressler, Dissertation 2000,
Med. Fakultt der Universitt
Mnchen
Determining Cannabinoids in
Blood Using ECNCI with
HP 5973 CI MSD
Harry Prest,
Agilent Publication Nr. 5967-6331
EI-Spectrum, Tetrahydrocannabinol Carboxcylic Acid, PFPA derivative, m/z 640; M+
PCI/CH4-Spectrum, Tetrahydrocannabinol Carboxcylic Acid, TMS derivative, m/z 489, 517, 529; [M + H]+, [M + C2H5]+, [M + C3H5]+
PCI/NH3-Spectrum, Tetrahydrocannabinol Carboxcylic Acid, TMS derivative, m/z 489; [M + H]+
106
ECNI/CH4-Spectrum, Tetrahydrocannabinol Carboxcylic Acid, TMS derivative, m/z 489; [M +H]-
PCI/CH4-Spectrum, Tetrahydrocannabinol Carboxcylic Acid, PFPA derivative, m/z 641, 669, 681; [M + H]+, [M + C2H5]+, [M + C3H5]+
PCI/NH3 -Spectrum, Tetrahydrocannabinol Carboxcylic Acid, PFPA derivative, m/z 641, 658; [M + H]+, [M + NH4]+
107
ECNI/CH4-Spectrum, Tetrahydrocannabinol Carboxcylic Acid, PFPA derivative, m/z 620, 640; [M HF]-, [M]-
SIM Mode, PFPA derivative

0.5pg/l each, Ions: m/z 620, 640
ECNI/CH4, Tetrahydrocannabinol Carboxcylic Acid
ECNI/NH3, Tetrahydrocannabinol Carboxcylic Acid
Ion Mode
Derivative
Concent.
Acquis.
Mode
EI
EI
PCI/CH4
PCI/NH3
NCI/CH4
PCI/CH4
PCI/NH3
NCI/CH4
NCI/CH4
NCI/NH3
TMS
PFPA
TMS
TMS
TMS
PFPA
PFPA
PFPA
PFPA
PFPA
10ng/l
10ng/l
10ng/l
10ng/l
10ng/l
10ng/l
10ng/l
10pg/l
0.5pg/l
0.5pg/l
Scan
Scan
Scan
Scan
Scan
Scan
Scan
Scan
SIM
SIM
Approximate
Signal/Noise
Ratio
650/1
620/1
40/1
80/1
10/1
140/1
75/1
15/1
40/1
60/1
Table: Tetrahydrocannabinol Carboxcylic Acid, Responses
108
trans Zearalenone
CAS-Nr. 17924-92-4
GC Parameters
Column: HP-5ms
30m x 0.25mm x 0.25m
Carrier Gas: Helium
Mode: Constant Flow
Oven Temp. Program
(4min)
MS Parameters
Mode: EI SCAN
Tune: Atune
Temperatures:
Mode: PCI/CH4 SCAN
Temperatures:
Mode: PCI/NH3 SCAN

Buffer Gas: Methane
Temperatures:
Remarks
Derivatization
a) Silylation (TMS) with BSTFA/
Z 2125) at a concentration of
10mg/ml in methanol is evaporated
with a gentle flow of nitrogen. To
the residue, 50l of derivatization
dissolved in ethyl acetate.
GC/MSD analysis.
EI-Spectrum, Zearalenone, underivatized, m/z 318; M+
109
Results
Underivatized analyte:
In EI scan mode measurements in
the concentrations range of 1ng
to 10ng are achievable. In PCI
mode, ammonia is the preferred
reagent gas.
TMS derivative:
The analyte reacts to make the
mono and di-derivative with a
response ratio of 0.4 : 1. PCI/NH3
responses are a factor of three
more sensitive compared to the
PCI/CH4.
PFPA derivative:
The di-derivative is preferentially
produced (90%) and elutes prior
to the mono-derivative. PCI/CH4 is
the preferred mode compared to
ammonia reagent gas.
In ECNI/CH4 mode the monoderivative dominates by a factor
of more than 10 to one. In SIM a
concentration of 0.5pg/l produces
approximately 30:1 signal:noise.
In addition to the mentioned
derivatization reactions,
pentafluorophenyl reaction was
tested and resulted in less
sensitive response when
compared to the PFPA data.
PCI/CH4-Spectrum, Zearalenone, underivatized, m/z 319, 347, 359; [M + H]+, [M + C2H5]+, [M + C3H5]+
PCI/NH3-Spectrum, Zearalenone, underivatized, m/z 319, 336; [M + H]+, [M+NH4]+
EI-Spectrum, Zearalenone, TMS mono-derivative, m/z 390; M+
110
EI-Spectrum, Zearalenone, TMS di-derivative, m/z 462; M+
PCI/CH4-Spectrum, Zearalenone, TMS mono-derivative, m/z 391, 419, 431; [M + H]+, [M + C2H5]+, [M + C3H5]+
PCI/CH4-Spectrum, Zearalenone, TMS di-derivative, m/z 463, 491, 503; [M + H]+, [M + C2H5]+, [M + C3H5]+
111
PCI/NH3-Spectrum, Zearalenone, TMS mono-derivative, m/z 391, 408; [M + H]+, [M+NH4]+
PCI/NH3-Spectrum, Zearalenone, TMS di-derivative, m/z 463 ; [M + H]+
EI-Spectrum, Zearalenone, PFPA mono-derivative, m/z 464; M+
112
EI-Spectrum, Zearalenone, PFPA di-derivative, m/z 610; M+
PCI/CH4-Spectrum, Zearalenone, PFPA mono-derivative, m/z 465, 493; [M + H]+, [M + C2H5]+
PCI/CH4-Spectrum, Zearalenone, PFPA di-derivative, m/z 611, 639, 651; [M + H]+, [M + C2H5]+, [M + C3H5]+
113
PCI/NH3-Spectrum, Zearalenone, PFPA mono-derivative, m/z 465, 482; [M + H]+, [M + NH4]+
PCI/NH3-Spectrum, Zearalenone, PFPA di-derivative, m/z 628; [M + NH4]+
ECNI/CH4-Spectrum, Zearalenone, PFPA mono-derivative, m/z 316, 444, 464; [M - 148(PFPA+H)]-, [M-HF]-, [M]-
114
ECNI/CH4-Spectrum, Zearalenone, PFPA di-derivative, molecular mass = 610 u m/z 463 ; [M-147]- (PFP = 147 u)
ECNI/CH4 SIM Mode
Zearalenone, PFPA mono-derivative, 200fg,

Retention Time: 10.68min, Ion: m/z 316;
Signal/Noise 30/1
115
The analysis of pharmacologically relevant compounds is carried out using different analytical
techniques. The combination of gas chromatography (GC) with mass spectrometry (MS)
is one of the most frequently applied because it offers high separating power, which is
advantageous in analyzing complex mixtures, and reliable identification of unknown compounds.
In the field of human and veterinarian medicine, drugs of abuse can be unambiguously
determined and accurately quantitated.
This brochure emphasizes the standard mass spectral technique of electron impact ionization
(EI) and both chemical ionization (CI) techniques; positive chemical ionization (PCI) and
electron capture negative ionization (ECNI or NCI). The data presented unmistakably indicates
CI is not merely a supplement to EI, but for most of the documented examples, improves
analytical results related to compound selectivity and detection sensitivity. To assist and
encourage the user in exploring CI, this brochure also presents an elementary understanding
of CI-theory and useful practical operating advice for CI on the Agilent MSD.
Data for 43 compounds are presented from a wide variety of drug classes; barbiturates,
benzodiazepines, -agonists, narcotics and steroids and others. All GC and MS method
parameters, derivatisation techniques, and the resulting EI/PCI/ECNI mass spectra are
documented. The results are briefly discussed for the different operating modes and reagent
gases applied. Attention is given to the quantitative data obtained especially in terms of
signal-to noise. The intention being to assist the user in choosing successful conditions and
modes to analyze samples for particular drugs at trace concentrations.
Copyright Agilent Technologies Company, 2001.

All rights reserved. Reproduction, adaption, or translation
without prior written permission is prohibited, except as
allowed under the copyright laws.
Printed in Germany
February, 2002
www.agilent.com/chem
Publication Number 5988-3921EN

EI and CI GC Ms

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

EI and CI GC Ms

Uploaded by

Copyright:

Available Formats

Analysis of

correct molecular mass. For

intermediate chemical agent.

2. Positive Chemical Ionization (PCI)

positioned just outside the

ability of the reaction partners to

The choice of the applied reagent

Ammonia has a high PA value

[M-H]+ = 269 m/z

Typical ammonia reactions

Figure 1. Total ion chromatograms of

Figure 2. Full Scan Spectra referring

3. Electron Capture Negative

consequently, the sensitivity. Low

Figure 3. Mass spectra of tetrahydrocannabinol

molecular structure and improve

has the advantages of almost

to the literature references and to

4.2 PCI and NCI Conditions

Care must be taken to insure that

Chemically aggressive reagents

connected to the injector and that

EI Spectrum, Acepromazine: m/z 326; M+

Mode: PCI/CH4 SCAN

PCI/NH3 Spectrum, Acepromazine: m/z 327; [M+H]+

EI-Spectrum, Amobarbital, underivatised, m/z 226; M+

EI-Spectrum, Amobarbital, PFBB-derivative, m/z 406; M+

concentration 20ng/l each, diluted in

ECNI/CH4-Spectrum, Amobarbital, PFBB-derivative: m/z 405; [M-H]-

EI-Spectrum, Barbital, underivatised, m/z 184; M+

EI-Spectrum, Barbital, PFBB-derivative, m/z 364; M+

ECNI/CH4-Spectrum, Barbital, PFBB-derivative: m/z 363; [M-H]-

EI-Spectrum, Butethal, underivatised, m/z 212; M+

EI-Spectrum, Butethal, PFBB-derivative, m/z 392; M+

ECNI/CH4-Spectrum, Butethal, PFBB-derivative: m/z 391; [M-H]-

EI-Spectrum, Pentobarbital, underivatised, m/z 226; M+

EI-Spectrum, Pentobarbital, PFBB-derivative, m/z 406; M+

ECNI/CH4-Spectrum, Pentobarbital, PFBB-derivative: m/z 405; [M-H]-

EI-Spectrum, Secobarbital, underivatised, m/z 238; M+

EI-Spectrum, Secobarbital, PFBB-derivative, m/z 418; M+

ECNI/CH4-Spectrum, Secobarbital, PFBB-derivative: m/z 417; [M-H]-

ECNI SIM, 5 Barbiturates

ECNI SIM, 5 Barbiturates, 1 pg/l each, see Table 1

EI-Spectrum, Alprazolam: m/z 308; M+

ECNI-Spectrum, Alprazolam: m/z 308; M-

EI-Spectrum, Bromazepam: m/z 315; M+

ECNI-Spectrum, Bromazepam: m/z 315; M-

EI-Spectrum, Diazepam: m/z 284; M+

ECNI-Spectrum, Diazepam: m/z 284; M-

EI-Spectrum, Flunitrazepam: m/z 313; M+

ECNI-Spectrum, Flunitrazepam: m/z 313; M-

EI-Spectrum, Triazolam: m/z 342; M+

ECNI-Spectrum, Triazolam: m/z 342; M-

ECNI SIM, Alprazolam

Table: Sensitivity Benzodiazepines

Tune: PCI-Methane Autotune

Gentle evaporation with nitrogen

EI-Spectrum, Benzoylecgonine, underivatised: m/z 289; M+

EI-Spectrum, Benzoylecgonine, TMS Derivative: m/z 361; M+

EI-Spectrum, Benzoylecgonine, PFPA Derivative, -O-CH-(CF3)2: m/z 439; M+

PCI/NH3-Spectrum, Benzoylecgonine, 10ng/l, TMS Derivative: m/z 362; [M + H]+

PCI/NH3-Spectrum, Benzoylecgonine, 50ng/l, TMS Derivative: m/z 362; [M+H]+

PCI/NH3-Spectrum, Benzoylecgonine, 10ng/l, PFPA Derivative, -O-CH-(CF3)2: m/z 440; [M+H]+